1. UOS di Milano Istituto di Ricerche Genetiche e Biomediche (IRGB)/CNR and and Humanitas Research Center Pavia, February 29 th, 2016 1

2. Induced pluripotent stem (iPS) cells iPSc are obtained by reprogramming of somatic cells through forced expression of few transcription factors. iPSc recapitulate embryonic stem cells characteristics such as self-renewal and ability to differentiate into the three germ layers. They represent a platform for disease modeling, drug screening and regenerative medicine. IRGB/CNR

3. Expression of stemness markers and pluripotency 3

4. iPSc produced osteoclasts From wt iPSc 10 X TRAP staining Dentine slice From oc/oc iPSc Osteoclast Differentiation Assay + M-CSF + RANKL Example of an osteoclast in culture Example of the actin ring staining From BAC iPSc The capacity to resorb dentine was completely rescued, demonstrating that BAC-mediated correction was successful

5. Cytogenetic characterization A further detailed cytogenetic analysis showed a small rearrangement involving the chromosome 18. Cl.7-1E5E14TG2a 5

6. Mesenchymal Stem Cells CNR/IRGB Mesenchymal Stem (or Stromal) Cells are a heterogeneous population of non-hematopoietic multipotent cells that can be isolated by adherence and expanded from a variety of tissues. They have the capacity to self-renew and differentiate into various mesodermal cell lineages (adipocytes, osteoblasts, chondrocytes) under specific culture conditions. Human MSCs CD34 - CD14 - CD45 - CD44 + CD71 + CD73 + CD90 + CD106 + Murine MSCs CD34 - CD11b - CD45 - CD9 + CD44 + Sca-1 +

7. Figure adapted from Seita and Weissman, 2010 and Medvinsky et al, 2011 Primitive hematopoietic stem cell c-Kit+ CD41+ Placenta Liver Yolk sac AGM region Umbilical cord Definitive hematopoietic stem cell c-Kit+ CD45low The murine hematopoietic system

8. Embryoid bodies formation 10X erythroidmyeloidmixed MΦ Gr Mo Er 10X 40X iPSc transfer into tissue culture treated plates + growth factors Colony-Forming Cells Assay on Methylcellulose FACS Analysis 8-20 days Hematopoietic cells CD41 CD45 Differentiation towards the hematopoietic lineage TRAP and Toluidine Blue stainings Multinucleated osteoclasts and resorption pits on dentine discs